Mutagenic analysis of AMP nucleosidase from Escherichia coli: Deletion of a region similar to AMP deaminase and peptide characterization by mass spectrometry

Kalla Kvalnes-Krick, James E. Labdon, Xiaoyan Ma, Edward Nieves, Vern L. Schramm

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

AMP nucleosidase (EC 3.2.2.4) from Escherichia coli and AMP deaminase (EC 3.5.4.6) from bakers' yeast are proposed to regulate cellular AMP levels under allosteric control of the activator ATP and the inhibitor, PO4. Both enzymes contain catalytic sites which bind AMP and regulatory sites which bind ATP. The deduced amino acid sequences of the proteins revealed only one region of homology in which six of eight amino acids are identical. A similar sequence is found in glyceraldehyde-3-phosphate dehydrogenase, phoE, ras proteins, RNA polymerase, K+-ATPase, nucleolin, and other proteins expected to have nucleotide or phosphate binding properties. In the crystal structure of glyceraldehyde-3-phosphate dehydrogenase, this sequence is part of the NAD+-binding site. The function of these amino acids was explored with a deletion mutant of AMP nucleosidase. The protein was over-produced in a pTZ construct using the AMP nucleosidase promoter which resulted in approximately 30% of the total protein as the desired enzyme. The mutation was characterized by DNA sequence analysis and by direct analysis of the peptides using high performance liquid chromatography-mass spectrometry. Deletion of amino acids 128-135, corresponding to DGSELTLD, produced an enzyme with a 20-fold decrease in Vmax but with smaller changes in substrate saturation kinetics, activation by MgATP, inhibition by inorganic phosphate, and inhibition by the tight-binding inhibitor, formycin 5-phosphate. The deletion mutant of AMP nucleosidase exhibits hysteresis in establishing a steady-state rate of product formation which is most pronounced in the absence of MgATP. These results establish that the sequence DGSELTLD in E. coli AMP nucleosidase is not required for binding of AMP, MgATP, or inorganic phosphate. However, the mutant enzyme has a structural defect related to the polymerization state which delays the onset of catalysis and decreases the catalytic efficiency.

Original languageEnglish (US)
Pages (from-to)8717-8726
Number of pages10
JournalJournal of Biological Chemistry
Volume268
Issue number12
StatePublished - Apr 25 1993

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AMP nucleosidase
AMP Deaminase
Escherichia coli
Mass spectrometry
Mass Spectrometry
Adenosine Triphosphate
Adenosine Monophosphate
Peptides
Amino Acids
Glyceraldehyde-3-Phosphate Dehydrogenases
Phosphates
Enzymes
Proteins
ras Proteins
DNA sequences
High performance liquid chromatography
DNA-Directed RNA Polymerases
NAD
Yeast
Catalysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mutagenic analysis of AMP nucleosidase from Escherichia coli : Deletion of a region similar to AMP deaminase and peptide characterization by mass spectrometry. / Kvalnes-Krick, Kalla; Labdon, James E.; Ma, Xiaoyan; Nieves, Edward; Schramm, Vern L.

In: Journal of Biological Chemistry, Vol. 268, No. 12, 25.04.1993, p. 8717-8726.

Research output: Contribution to journalArticle

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abstract = "AMP nucleosidase (EC 3.2.2.4) from Escherichia coli and AMP deaminase (EC 3.5.4.6) from bakers' yeast are proposed to regulate cellular AMP levels under allosteric control of the activator ATP and the inhibitor, PO4. Both enzymes contain catalytic sites which bind AMP and regulatory sites which bind ATP. The deduced amino acid sequences of the proteins revealed only one region of homology in which six of eight amino acids are identical. A similar sequence is found in glyceraldehyde-3-phosphate dehydrogenase, phoE, ras proteins, RNA polymerase, K+-ATPase, nucleolin, and other proteins expected to have nucleotide or phosphate binding properties. In the crystal structure of glyceraldehyde-3-phosphate dehydrogenase, this sequence is part of the NAD+-binding site. The function of these amino acids was explored with a deletion mutant of AMP nucleosidase. The protein was over-produced in a pTZ construct using the AMP nucleosidase promoter which resulted in approximately 30{\%} of the total protein as the desired enzyme. The mutation was characterized by DNA sequence analysis and by direct analysis of the peptides using high performance liquid chromatography-mass spectrometry. Deletion of amino acids 128-135, corresponding to DGSELTLD, produced an enzyme with a 20-fold decrease in Vmax but with smaller changes in substrate saturation kinetics, activation by MgATP, inhibition by inorganic phosphate, and inhibition by the tight-binding inhibitor, formycin 5-phosphate. The deletion mutant of AMP nucleosidase exhibits hysteresis in establishing a steady-state rate of product formation which is most pronounced in the absence of MgATP. These results establish that the sequence DGSELTLD in E. coli AMP nucleosidase is not required for binding of AMP, MgATP, or inorganic phosphate. However, the mutant enzyme has a structural defect related to the polymerization state which delays the onset of catalysis and decreases the catalytic efficiency.",
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